CA11 Antibody

What is CA11 and Why is it Important in Neuroscience Research?

CA11 (Carbonic Anhydrase XI), also known as CARP2 or CARP XI, belongs to the alpha-carbonic anhydrase family. Unlike other carbonic anhydrases, CA11 does not have catalytic activity but plays significant roles in central nervous system function. CA11 is observed in multiple tissues including the cerebellum, cerebrum, liver, stomach, small intestine, colon, kidney, and testis, with particularly prominent expression in the Purkinje cells of the cerebellum .

CA11 contains a hydrophobic N-terminal region that serves as a signal sequence and includes asparagine glycosylation sites. Research indicates that CA11 functions as a secreted synaptic protein that can act as a neurexin ligand. Recent studies have shown that CA11 secreted by depolarized neurons can inhibit glioma cell growth, suggesting its potential role in regulating neuronal activity-dependent processes .

What Applications Are CA11 Antibodies Validated For?

CA11 antibodies have been validated for multiple applications, with specific recommended dilutions and protocols for each application. The choice of application depends on your research question and the specific cellular or molecular aspects of CA11 you wish to investigate.

When selecting an application, consider that Western blotting is ideal for quantifying total protein levels, IHC for tissue localization patterns, IF for subcellular localization, FC for quantifying CA11 in specific cell populations, and ELISA for detecting secreted CA11 in conditioned media or biological fluids .

How Should CA11 Antibodies Be Stored and Handled to Maintain Optimal Activity?

Proper storage and handling of CA11 antibodies is critical for maintaining their specificity and sensitivity in experimental applications. Research demonstrates that antibody performance can significantly degrade with improper handling.

Recommended Storage Conditions:

• Store at -20°C for long-term storage (stable for one year after shipment)

• For frequent use, store small aliquots at 4°C for up to one month

• Avoid repeated freeze/thaw cycles as they can denature antibodies and reduce activity

• Most CA11 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol pH 7.3

• Some preparations (20μL sizes) may contain 0.1% BSA for additional stability

Handling Best Practices:

• Briefly centrifuge vials before opening to collect all material at the bottom

• When aliquoting, use sterile microcentrifuge tubes and maintain sterile technique

• Aliquot volumes appropriate for single experiments to avoid freeze/thaw cycles

• Allow antibodies to reach room temperature before opening to avoid condensation

• Do not vortex antibodies; mix by gentle inversion or flicking

When prepared and stored properly, most CA11 antibodies maintain their reactivity for at least 12 months from the date of receipt .

What Are the Key Considerations for Validating CA11 Antibody Specificity?

Antibody validation is critical for ensuring experimental reproducibility and reliable results. For CA11 antibodies, several validation strategies are recommended based on current research standards:

Recommended Validation Methods:

• Genetic Controls: The optimal validation method employs wild-type cells alongside CRISPR knockout cells. This rigorous approach allows for definitive confirmation of antibody specificity .

• Expression Controls: Test antibodies on CA11-transfected vs. non-transfected cell lines (e.g., 293T cells) . Expected bands should appear at approximately 36-43 kDa in transfected cells but not in non-transfected controls.

• Peptide Competition Assay: Pre-incubate the antibody with the immunizing peptide before application to sample. Signal elimination confirms specificity.

• Multi-application Validation: Verify consistent results across different applications (WB, IHC, IF) to confirm target recognition regardless of protein conformation.

• Cross-reactivity Testing: Test against related carbonic anhydrase family members to ensure specificity within this protein family.

Validation Data Analysis:

• For WB: Evaluate band pattern, molecular weight (36-48 kDa depending on glycosylation), and signal-to-noise ratio

• For IHC/IF: Compare staining pattern with known expression profile in tissues (high in cerebellum, particularly Purkinje cells)

• For FC: Validate with known CA11-expressing cells (positive) and non-expressing cells (negative)

A comprehensive validation strategy increases confidence in antibody specificity and experimental results .

How Can I Optimize Western Blot Protocols for CA11 Detection?

Western blot optimization for CA11 detection requires attention to several key parameters to ensure specific and sensitive detection of this 36 kDa protein. CA11 can appear at different molecular weights (36-48 kDa) due to post-translational modifications, particularly glycosylation.

Sample Preparation:

• Use fresh tissue/cells when possible

• For brain tissue samples, rapid extraction and processing are essential to prevent protein degradation

• Recommended lysis buffer: RIPA buffer supplemented with protease inhibitors

• Add deglycosylation enzymes (PNGase F) to a portion of your sample to confirm glycosylation-related band shifts

Electrophoresis and Transfer Parameters:

• Use 10-12% polyacrylamide gels for optimal resolution of CA11 (36 kDa)

• Run at 100-120V to prevent overheating

• Transfer to PVDF membranes (preferred over nitrocellulose for glycoproteins) at 100V for 1 hour or 30V overnight at 4°C

Antibody Incubation:

• Block with 5% non-fat milk or 3% BSA in TBST

• Optimal antibody dilutions: 1:500-1:2000 for most CA11 antibodies

• Incubate primary antibody overnight at 4°C for best results

• Secondary antibody: Anti-rabbit or anti-mouse HRP (depending on primary antibody host) at 1:2500-1:5000

Detection Optimization:

• Use ECL substrate appropriate for expected expression level

• For low expression, enhanced chemiluminescent substrates are recommended

• Expected bands: 36 kDa (main band), with possible additional bands at 40-48 kDa (glycosylated forms)

If multiple bands are observed, validation experiments with deglycosylation enzymes can help confirm which bands represent CA11 isoforms versus non-specific binding .

What Controls Should Be Included in CA11 Antibody Experiments?

Proper controls are essential for interpreting CA11 antibody experimental results accurately. Based on rigorous research practices, the following controls should be included:

Positive Controls:

• Human kidney tissue lysate (confirmed to express CA11)

• U2OS cells (for IF experiments)

• A431 cells (for flow cytometry)

• CA11-transfected 293T cells

Negative Controls:

• Primary antibody omission control (tissue/cells treated with blocking solution and secondary antibody only)

• Isotype control (matching the primary antibody's host species and isotype)

• Non-transfected 293T cells (for transfection experiments)

• Ideally, CA11 knockout/knockdown cells or tissues (CRISPR-edited cells)

Validation Controls:

• Peptide competition/blocking control (pre-incubation of antibody with immunizing peptide)

• Cross-reactivity controls (testing on tissues known to lack CA11 expression)

• For IHC, include antigen retrieval controls with and without TE buffer pH 9.0 treatment

Loading Controls:

• For Western blots: β-actin, GAPDH, or other housekeeping proteins

• For IF/IHC: Nuclear stain (DAPI/Hoechst) and cell type-specific markers

Including these controls in your experimental design allows for confident interpretation of results and helps troubleshoot potential issues with antibody specificity or experimental conditions .

What Are the Best Practices for Immunohistochemistry Using CA11 Antibodies?

Successful immunohistochemical detection of CA11 requires optimization of several key parameters. Based on published methodologies, the following best practices are recommended:

Tissue Preparation:

• Fixation: 4% paraformaldehyde for 24-48 hours is optimal for brain tissue

• Processing: Standard paraffin embedding with careful attention to temperature to avoid antigen degradation

• Sectioning: 5-7 μm thickness is ideal for CA11 detection

Antigen Retrieval (Critical Step):

• Primary method: TE buffer pH 9.0 (recommended for most CA11 antibodies)

• Alternative method: Citrate buffer pH 6.0

• Heat-induced epitope retrieval: 95-98°C for 15-20 minutes followed by gradual cooling

Blocking and Antibody Incubation:

• Block with 5-10% normal serum from secondary antibody host species

• Add 0.1-0.3% Triton X-100 for membrane permeabilization

• CA11 antibody dilution: 1:20-1:200 (optimize for each specific antibody)

• Incubation time: Overnight at 4°C yields best results

Detection Systems:

• DAB (3,3'-diaminobenzidine) for brightfield microscopy

• Fluorescent secondary antibodies for co-localization studies

Validated Tissues for Positive Control:

• Human brain tissue (especially cerebellum for Purkinje cells)

• Human lung tissue

• Human ovary tissue

• Human placenta tissue

• Human spleen tissue

• Human testis tissue

For dual labeling experiments, CA11 can be co-stained with neuronal markers to study its distribution in specific neuronal populations. Careful titration of antibody concentration is essential for optimal signal-to-noise ratio .

How Can I Use CA11 Antibodies to Study Its Role in Neuronal-Glioma Interactions?

Recent research has revealed that CA11 plays a significant role in neuronal-glioma interactions, specifically as a negative regulator of neuronal activity-dependent glioma growth. The following methodological approaches can be used to study this phenomenon:

Co-culture Experimental Design:

• Establish primary neuronal cultures from rat/mouse cortex

• Treat neurons with high KCl (50mM) to induce depolarization and CA11 secretion

• Collect conditioned medium (CM) and concentrate using ultrafiltration

• Apply CM to glioma cell lines (U251, U87) with or without CA11 immunodepletion

• Measure proliferation using MTT assay or BrdU incorporation

Key Experimental Groups:

• Control medium (from non-depolarized neurons)

• CM (from depolarized neurons containing secreted CA11)

• CM with CA11 immunodepleted

• CM with control IgG immunodepleted

• Recombinant CA11 treatment

Analytical Methods:

• Western blot to confirm CA11 secretion and immunodepletion

• qRT-PCR to measure CA11 mRNA expression in glioma cells after CM treatment

• Luciferase assays with CA11 promoter constructs to assess transcriptional regulation

• Pathway analysis using Akt inhibitors to elucidate signaling mechanisms

Research Findings:
Research has shown that CM from depolarized neurons contains increased levels of CA11, which inhibits glioma cell proliferation. When CA11 is depleted from CM, glioma proliferation increases significantly. Additionally, CM treatment reduces CA11 expression in glioma cells through the Akt signaling pathway .

These findings suggest that CA11 functions as a tumor suppressor in gliomas, and its expression is negatively associated with glioma grade and patient survival .

What Approaches Can Be Used to Study CA11 in Flow Cytometry Applications?

Flow cytometry offers a powerful approach for quantifying CA11 expression in different cell populations and studying its regulation under various conditions. For optimal results with CA11 antibodies in flow cytometry, consider the following methodological guidelines:

Sample Preparation Protocol:

• Isolate cells of interest (primary cells or cell lines)

• For intracellular staining (most common for CA11):

  • Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.1% saponin or 0.1% Triton X-100 in PBS

  • Block with 2% BSA in PBS for 30 minutes

• For surface CA11 detection (if studying externalized protein):

  • Use non-fixed cells

  • Block with 2% BSA in PBS for 30 minutes

Antibody Staining:

• Use anti-CA11 antibody at recommended concentration (typically 0.25 μg per 10^6 cells in 100 μl suspension)

• Incubate for 30-60 minutes at room temperature or 4°C

• Wash 3× with PBS containing 0.5% BSA

• Apply fluorophore-conjugated secondary antibody appropriate for your instrument configuration

• For multicolor applications, include panels with lineage markers

Controls:

• Unstained cells

• FMO (Fluorescence Minus One) controls

• Isotype control antibody

• Positive control: A431 cells (validated for CA11 detection)

• Negative control: Jurkat T cells (do not express CD11d)

Analysis Considerations:

• Gate on viable cells using appropriate viability dye

• Set compensation using single-stained controls

• When studying neuroinflammatory conditions, consider pairing CA11 with markers of cell activation

• For clinical samples, markers to distinguish cell subpopulations (e.g., CD14/CD16 for monocyte subsets) are essential

Flow cytometry data has revealed that CA11 expression varies across monocyte subpopulations, with CD14+CD16+ nonclassical monocytes showing the highest surface expression levels .

How Can I Quantify CA11 Protein Expression in Research Applications?

Accurate quantification of CA11 expression is essential for understanding its biological role in normal and pathological conditions. Multiple methods can be employed, each with specific advantages for different research questions:

Western Blot Quantification:

• Use recombinant CA11 protein to create a standard curve (5-100 ng range)

• Load equal amounts of total protein (confirmed by BCA/Bradford assay)

• Include housekeeping controls (β-actin, GAPDH)

• Use densitometry software (ImageJ, Image Lab) to quantify band intensity

• Normalize CA11 signal to loading control

• For glycosylated forms, consider the sum of all specific bands (36-48 kDa)

ELISA-Based Quantification:

• Commercial CA11 ELISA kits typically have detection ranges of 0.1-10 ng/ml

• For secreted CA11 in conditioned media or cerebrospinal fluid

• Allows high-throughput analysis of multiple samples

• Recommended dilution for antibodies: 1:5000-1:20000

Flow Cytometry Quantification:

• Measure mean fluorescence intensity (MFI) as indicator of protein abundance

• Use calibration beads with known antibody binding capacity

• Convert MFI to molecules of equivalent soluble fluorochrome (MESF)

Immunohistochemical Quantification:

• Semi-quantitative scoring (0-3+) based on staining intensity

• Digital image analysis using software like QuPath or ImageJ

• H-score calculation (percentage of positive cells × intensity)

Comparison of Quantification Methods:

When reporting CA11 expression data, clearly specify the quantification method, normalization approach, and statistical analysis to ensure reproducibility .

What is Known About CA11's Role in Glioma Pathogenesis and How Can Antibodies Help Study This?

CA11 has emerged as a significant player in glioma biology, with research indicating it functions as a tumor suppressor. CA11 antibodies are valuable tools for investigating this role through various methodological approaches:

CA11 Expression in Gliomas:

• CA11 expression is reduced in clinical glioma samples compared to normal brain tissue

• Expression levels correlate negatively with histological grade

• Low CA11 expression is associated with shorter survival in multiple independent datasets:

  • Repository of Brain Neoplasia Data (REMBRANDT)

  • The Cancer Genome Atlas (TCGA) Lower Grade Glioma (LGG)

  • GEO datasets (GSE4271 and GSE42669)

Functional Studies Using CA11 Antibodies:

• Immunoprecipitation of Secreted CA11:

  • Use anti-CA11 antibodies to deplete CA11 from neuronal conditioned medium

  • Apply to glioma cells to assess proliferation effects

  • Results show CA11 depletion enhances glioma cell proliferation

• CA11 Knockdown Studies:

  • Validate knockdown efficiency using CA11 antibodies in Western blot

  • Effects of CA11 knockdown in glioma models:

    • Promoted cell growth and clone formation

    • Enhanced migration

    • Inhibited apoptosis

    • Increased tumor size in xenografted nude mice

• Signaling Pathway Analysis:

  • Use CA11 antibodies to monitor protein levels after pathway inhibition

  • Research shows neuronal CM inhibits CA11 expression in glioma cells via Akt signaling

Survival Analysis Based on CA11 Expression:

These findings suggest CA11 as a potential therapeutic target and prognostic marker for gliomas. CA11 antibodies are essential tools for validating expression patterns in patient samples and functional studies in experimental models .

How Can CA11 Antibodies Be Used in Comparative Studies Across Different Species?

CA11 is evolutionarily conserved across mammals, making comparative studies valuable for understanding its fundamental biological functions. When using CA11 antibodies for cross-species research, consider the following methodological approaches:

Sequence Homology Analysis:

• Human and mouse CA11 share approximately 95% amino acid sequence identity

• Human and rat CA11 share approximately 94% amino acid sequence identity

• Specific regions may have higher conservation, affecting antibody cross-reactivity

Cross-Species Validation Approaches:

• Western Blot Comparison:

  • Run samples from multiple species side-by-side

  • Compare band patterns and molecular weights

  • Human CA11: ~36 kDa primary band

  • Mouse/Rat CA11: ~36-38 kDa (slight variations in glycosylation)

• IHC/IF Optimization by Species:

  • Antigen retrieval conditions may differ between species

  • Antibody dilutions often require species-specific optimization

  • Fixation protocols may need adjustment (particularly for rodent tissues)

• Control Samples by Species:

  • Human: Cerebellum (Purkinje cells), kidney

  • Mouse: Cerebellum, kidney, testis

  • Rat: Cerebellum, kidney, testis

Application Examples:

• Comparative neuroanatomical studies of CA11 distribution across species

• Evaluation of CA11 in animal models of glioma and neurological diseases

• Evolutionary studies of carbonic anhydrase family member functions

What Are the Challenges in Detecting Post-Translational Modifications of CA11?

CA11 undergoes several post-translational modifications (PTMs), particularly glycosylation, which can significantly impact protein detection and function. Understanding and addressing these challenges is critical for accurate CA11 research:

Key CA11 Post-Translational Modifications:

• N-glycosylation: CA11 contains asparagine glycosylation sites that contribute to its secretion and stability

• Signal peptide cleavage: The hydrophobic N-terminal region serves as a signal sequence that is cleaved during secretion

• Potential phosphorylation sites that may regulate function

Challenges in PTM Detection:

• Variable molecular weight: Glycosylation can shift CA11's apparent molecular weight from 36 kDa (unmodified) to 40-48 kDa in Western blots

• Epitope masking: Glycans may obscure antibody binding sites, affecting detection efficiency

• Tissue-specific glycosylation patterns: The extent and type of glycosylation may vary between tissues

• Species differences: Glycosylation patterns can differ between human, mouse, and rat CA11

Methodological Solutions:

• Enzymatic deglycosylation:

  • Treat protein samples with PNGase F to remove N-linked glycans

  • Compare migration patterns before and after treatment

  • Run treated and untreated samples side-by-side in Western blots

• PTM-specific antibodies:

  • Use antibodies that specifically recognize glycosylated or non-glycosylated forms

  • Develop antibodies against specific PTM sites

• Mass spectrometry approaches:

  • Liquid chromatography-tandem mass spectrometry (LC-MS/MS)

  • Glycopeptide enrichment strategies

  • Site-specific PTM mapping

• Expression systems for comparison:

  • Bacterial expression (no glycosylation)

  • Mammalian expression systems (physiological glycosylation)

  • Comparison of recombinant and native proteins

These methodological approaches can help distinguish between different CA11 forms and understand how PTMs affect its function in neuronal-glial interactions and potential roles in pathology .

How Can CA11 Antibodies Be Used to Study the Secretion and Extracellular Functions of CA11?

CA11 functions as a secreted protein that mediates intercellular communication, particularly in neuronal-glial interactions. The following methodological approaches utilize CA11 antibodies to investigate its secretion and extracellular functions:

Detecting Secreted CA11:

• Conditioned Media Analysis:

  • Culture neurons or other CA11-expressing cells

  • Induce depolarization with high KCl (50mM) medium

  • Collect and concentrate conditioned media using ultrafiltration

  • Analyze by Western blot using CA11 antibodies

  • Research shows depolarized neurons secrete significantly higher levels of CA11 compared to non-depolarized neurons

• ELISA Quantification:

  • Develop sandwich ELISA using capture and detection antibodies

  • Quantify CA11 in biological fluids (CSF, serum) or conditioned media

  • Optimal antibody dilutions: 1:5000-1:20000

Functional Analysis of Secreted CA11:

• Immunodepletion Studies:

  • Deplete CA11 from conditioned media using specific antibodies

  • Apply depleted media to target cells (e.g., glioma cell lines)

  • Measure biological effects (proliferation, migration, signaling)

  • Research shows CA11 depletion enhances glioma cell proliferation

• Recombinant CA11 Application:

  • Produce HA-tagged recombinant CA11 in expression systems

  • Purify and apply to target cells

  • Detect binding using anti-HA or anti-CA11 antibodies

  • Analyze downstream effects on cellular function

• Receptor Identification:

  • Use tagged CA11 for pull-down assays to identify binding partners

  • Perform crosslinking studies followed by immunoprecipitation

  • Research indicates CA11 functions as a neurexin ligand

Visualization of CA11 Secretion and Binding:

• Live-cell imaging with fluorescently tagged CA11

• Immunofluorescence to detect bound CA11 on target cells

• Co-localization studies with potential receptors

These approaches have revealed that CA11 secreted by neurons acts as a negative regulator of glioma growth, providing insights into its potential therapeutic applications in brain tumors .

What Are the Most Advanced Research Applications for CA11 Antibodies in Neuroscience?

CA11 antibodies are being employed in cutting-edge neuroscience research to elucidate its roles in brain function and disease. Here are the most advanced applications currently being pursued:

Single-Cell Multi-Omics Analysis:

• Integration of CA11 protein detection with transcriptomics at single-cell level

• CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) approaches

• Correlation of CA11 expression with cell-type-specific markers

• Identification of CA11-expressing cell populations in complex tissues

In Vivo Imaging and Functional Studies:

• Development of fluorescently labeled CA11 antibodies for in vivo imaging

• PET imaging with radiolabeled antibodies to track CA11 expression in animal models

• Correlation of CA11 dynamics with neuronal activity and disease progression

Therapeutic Development and Target Validation:

• Use of CA11 antibodies to modulate CA11 function in disease models

• Validation of CA11 as a potential therapeutic target for gliomas

• Low CA11 expression correlates with poor survival in glioma patients across multiple datasets

• CA11 knockdown promotes glioma progression in xenograft models

Neuronal Activity-Dependent Regulation:

• Study of how neuronal activity regulates CA11 expression and secretion

• Investigation of CA11's role in modulating synaptic function

• Exploration of CA11 as a neurexin ligand affecting synaptic organization

Brain Tumor Microenvironment Research:

• Analysis of CA11 in neuron-glioma crosstalk

• In situ detection of CA11 in tumor microenvironments

• CA11 as part of a gene signature associated with radiotherapy response and prognosis in gliomas

• Contrasting functions with other neuron-derived factors like neuroligin-3 that promote glioma growth

These advanced applications are expanding our understanding of CA11's multifaceted roles in the nervous system and its potential as a therapeutic target for brain tumors and possibly other neurological disorders.

How Can Researchers Troubleshoot Common Issues with CA11 Antibody Applications?

When working with CA11 antibodies, researchers may encounter various technical challenges. Here are methodological approaches to troubleshoot common issues:

Western Blot Troubleshooting

Immunohistochemistry/Immunofluorescence Troubleshooting

ELISA Troubleshooting